Proteins and polypeptides play an important role in a variety of applications. For example, proteins and polypeptides can be employed as research compounds, drug candidates, and in other therapeutic applications. One of the more significant challenges in the development of these and other applications is the development of cost effective and efficient processes for purification of proteins and polypeptides, particularly on a commercial scale. It is also challenging to develop feasible methods of producing proteins of suitable purity and amounts for research purposes as well. While many methods are now available for large-scale production of proteins, crude preparations contain not only the desired product but also closely related impurities that are difficult to separate from the desired product. Moreover, biological sources of proteins usually produce complex mixtures of materials.
Procedures for purification of proteins from cell debris can initially depend on the site of expression of the protein. Some proteins can be engineered so that they are secreted directly from the cell into the surrounding growth media; others are retained within the cell. For the latter proteins, the first step of a purification process involves lysis of the cell, which can be done by a variety of methods, including mechanical shear, osmotic shock, or enzymatic treatments. Such disruption releases the entire contents of the cell into the homogenate and, in addition, produces subcellular fragments that can be difficult to remove due to their small size. These are generally removed by differential centrifugation or by filtration, leading to a clarified solution. The same problem arises, although on a smaller scale, with directly secreted proteins due to the natural death of cells and release of intracellular host cell proteins in the course of the protein production run.
Once a clarified solution containing a protein of interest has been obtained, its separation from the other proteins produced by the cell is usually attempted using a combination of various chromatography techniques. These techniques separate mixtures of proteins on the basis of their charge, degree of hydrophobicity, size, or affinity, to name but a few separation criteria. Several different chromatography resins are available for each of these techniques, allowing accurate tailoring of the purification scheme to the particular protein involved.
The essence of each of these separation methods is that proteins can be caused either to move at different rates down a long column, achieving a physical separation that increases as they pass further down the column, or to adhere selectively to the separation medium, being then differentially eluted by different solvents. Alternatively, the separation can be based on the association of a protein with a column matrix material and thus depends on the sample not moving down the column until it is eluted therefrom. In some cases, the desired protein is separated from impurities when the impurities specifically adhere to the column and the protein of interest does not; that is, the protein of interest is present in the “flow-through”.
Affinity chromatography and hydrophobic interaction chromatography techniques have been developed to supplement the more traditional size exclusion and ion exchange chromatographic protocols. Affinity chromatography relies on the interaction of a target protein with an immobilized ligand. The ligand can be specific for the particular protein of interest in which case the ligand can be, for example, a substrate, substrate analog, inhibitor, or antibody. Alternatively, the ligand can be adapted to react with another protein. General ligands, such as adenosine monophosphate (AMP), adenosine diphosphate (ADP), nicotine adenine dinucleotide (NAD), or certain dyes can be employed to recover one or more classes of proteins.
In a common affinity chromatography isolation scheme, a specific interaction between an insoluble immobilized ligand and a soluble target protein can be advantageously employed (see generally, Janson & Rydén (eds), (1998) Protein Purification: Principles, High Resolution Methods, and Applications (2nd ed.), Wiley-Liss, New York; Johnstone & Thorpe, (1987) Immunochemistry in Practice, (2nd ed.), Blackwell Scientific Publications, pp. 207-240). By interacting with the ligand, the target protein is temporarily rendered insoluble and is retained on the solid support on which the ligand is immobilized while the soluble contaminants are eluted. The binding of the target protein to the ligand conventionally takes place in an aqueous buffer at a neutral pH. The target protein is subsequently released from the immobilized ligand by a change in the elution conditions, such as a change in the pH; an increase in temperature; elution with a denaturing agent, an organic solvent, or an unphysiologically high concentration of a salt; or elution with a compound that competes for a binding site on the target protein. As a result of these procedures, the target protein is often recovered in a denatured form and must be subjected to further manipulations in order to become re-folded into its native conformation.
Examples of commonly employed ligands are antibodies, in particular monoclonal antibodies (Mabs), which can be made to be more selective and to bind more firmly than most other known ligands. As a result, monoclonal antibodies can result in a higher purity of the eluted protein product. In order to obtain an antibody in sufficient quantities, however, the protein to be purified usually must be available in substantially pure form for the immunization procedure. Often, this is an insurmountable limitation.
Colorimetric methods are often based on a primary and secondary antibody-conjugate system. Antibodies have the advantage of being very specific and sensitive. However, antibody-based methods also have the potential for non-specific interactions due to antibody and antibody-conjugate adsorption to the peptide library itself. These two-step methods also consist of more variables than simple one-step methods, thus requiring additional optimization. With direct fluorescent detection methods, autofluorescence of the resin beads can be a major drawback, depending on the type of resin used. Thus, antibody-based approaches to protein purification and detection can be cumbersome and nonspecific.
Radiological techniques have also been employed in protein purification schemes. In these approaches, a protein is labeled with a detectable radioactive moiety. Disadvantages of these radiological techniques include the need to handle hazardous radioactive material, radiolysis of the labeled protein, and the potential structural modification of target protein due to radiolabeling. Exposure times can also be a limiting factor.
Additionally, most of these methods are not appropriate for the large-scale production of a target protein, since they are inefficient in target protein recovery or are only partially effective in removing impurities. Large scale purification methods which employ immunoaffinity chromatography (see e.g., Wallen et al. (1983) Eur. J. Biochem. 133: 681-686) are limited by the cost of antibody resins, the difficulty associated with sterilizing these resins, and by the potential for the antibodies, or fragments thereof, to contaminate the recovered target protein. Radiological methods require the use of radioactivity, which, as disclosed hereinabove, can be undesirable. All of the methods discussed suffer from a lack of specificity. Furthermore, fusion proteins can require the fusion of a target protein with a sequence that can be longer than that of the target protein, or alternatively, can interfere with the activity of the target protein. In cases where the retention of the biological activity of the protein is essential, the removal of the fused moiety would be necessary, as well as the purification of the target protein from the fused moiety, which can result in drastically reduced yields.
Therefore, the need for a cost-effective affinity ligand to purify target proteins remains. In order to obtain a high degree of purity, a ligand with a high avidity towards a target protein is needed. Additionally, there is a concurrent need for a short tag that can associate with the ligand. Such a tag can be associated with a target protein sequence to aid in purification and/or detection of the target protein. Preferably, the tag is short enough that it does not interfere with the structure or function of the target protein. The problem then, is to identify a ligand with a high avidity for a short tag, yet without such high avidity that the target protein cannot be disassociated from the ligand without denaturation. The present invention solves this and other problems associated with protein purification and detection.